Culture having phenol oxidase-like activity

ABSTRACT

It is intended to provide means of efficiently, economically and conveniently dyeing fibers or hair, bleaching pulp or fibers, removing phenol compounds from liquid wastes, degrading endocrine disruptors, producing phenolic resins, producing artificial lacquer coatings, improving wood qualities, etc. A culture of a strain belonging to the genus  Flammulina ; a culture originating in the above strain which is obtained by culturing the strain at a pH value exceeding 7 and has a phenol oxidase-like activity; a process for producing the culture; a dyeing method which comprises contacting a subject to be dyed with a dye in the presence of the above culture; and a dyeing composition containing the above culture.

TECHNICAL FIELD

The present invention relates to a culture having phenol oxidase-likeactivity and a method for producing the culture, a staining method and astaining composition. More specifically, the present invention relatesto a culture useful in staining of fiber and hair, bleaching of pulp andfiber, removal of a phenolic compound or the like in waste liquor,degradation of endocrine disruptors, preparation of a phenolic resin,production of artificial lacquer, improvement in woody properties andthe like, a production method, capable of obtaining the cultureconveniently and inexpensively in a large amount, a method for stainingfibers, hair or the like, capable of staining with various dyes, and astaining composition useful for such staining.

BACKGROUND ART

Oxidases showing oxidization actions on various substrates such asphenolic compounds and polyphenolic compounds can be mainly classifiedinto two groups of peroxidases and phenol oxidases.

The above-mentioned peroxidases catalyze oxidation of various substratesand require the presence of hydrogen peroxide as a common substrate in areaction system. On the other hand, the phenol oxidases catalyzeoxidation of various substrates and require the presence of molecularoxygen as a common substrate. Therefore, among known oxidases, since theabove-mentioned phenol oxidases can catalyze oxidation of varioussubstrates in the presence of oxygen in the air, the phenol oxidases aresuitable for diversified chemical reactions such as coloring,decolorization, polymerization and degradation caused by generation ofradical species as intermediates in the presence of oxygen.

The above-mentioned phenol oxidases have various catalytic abilitiesbased on the action of radical species formed as reaction intermediates.For instance, it has been disclosed that when phenothiazine-10-propionicacid is used as a mediator, the degradation reaction of indigo can beefficiently carried out with laccase, which is a phenol oxidase (2001,University of Fukui, Center for Cooperative Research in Science andTechnology, “Materials on Workshop for High-Technology,” p. 55).

The above-mentioned oxidases have conventional been found in variousplants, bacteria, fungi and the like. For instance, in the plants, theabove-mentioned oxidases have been found in secretory pipe ofAnacardiaceae, peaches, chestnuts, and those belonging to Podocarpaceae.In the fungi, the above-mentioned oxidases have been found inAspergillus, Botrytis, Myrothecium, Penicillium, Pestalotia, Rhizoctoniaand the like belonging to the genus Deuteromycotina; Pleurotus,Lentinus, Polyporus, Trametes, Coriolus and the like belonging to thegenus Basidiomycotina; Podospora, Neurospora, Monocillium and the likebelonging to the genus Ascomycotina. In the bacteria, theabove-mentioned oxidases have been found in Bacillus, Azospirillum,Streptomyces, Aerobacter and the like. In addition, in the ediblemushrooms, which are Basidiomysetes, the above-mentioned oxidases havebeen found in, for instance, Schizophyllum commune, Coriolus versicolor,Pycnoporus coccineus, Pleurotus octreatus, Fomitella fraxinea and thelike.

However, since many phenol oxidases possess the optimum pH near anacidic pH due to their limited uses. In addition, phenol oxidases havingthe optimum pH in the neutral to alkaline pH may not efficiently act onvarious substrates near the neutral pH because their optimum pHs aregreatly changed depending upon the substrates used.

On the other hand, Flammulina velutipes has been found to exhibitlaccase activity by culturing Flammulina velutipes in a medium at a pHof 6.0 (Japanese Patent Laid-Open No. Sho 60-156385).

However, the above-mentioned laccase is an enzyme having the optimum pHon the acidic pH, and has a defect that the activity is lowered in a pHof the neutral to alkaline region.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a culture of a strainbelonging to the genus Flammulina, which can be used in reaction ofvarious compounds, particularly phenolic compounds, aminophenoliccompounds and diaminophenolic compounds as substrates, and is excellentin applicability to efficient staining of fiber and hair, bleaching ofpulp and fiber, removal of a phenolic compound in waste liquor,degradation of endocrine disruptors, preparation of phenolic resin,production of artificial lacquer, and improvement in woody properties.Also, an object of the present invention is to provide a method ofproducing the culture, capable of obtaining the above-mentioned cultureeasily and inexpensively in a large amount. Further, an object of thepresent invention is to provide a staining method, capable of stainingefficiently and easily with various dyes, specifically phenoliccompounds, aminophenolic compounds, diaminophenolic compounds, naturallyoccurring materials (flavonoids and the like), black-pigmentconstituents in animals and plants, and the like. In addition, an objectof the present invention is to provide a staining composition which iseasily handled and is capable of staining with various dyes,specifically phenolic compounds, aminophenolic compounds,diaminophenolic compounds, naturally occurring materials (flavonoids andthe like), black-pigment constituents in animals and plants, and thelike.

The summary of the present invention is:

-   [1] a culture from a strain belonging to the genus Flammulina,    wherein the culture has phenol oxidase-like activity and at least    one substrate specificity selected from the group consisting of    {circle around (1)} to {circle around (0)}:-   {circle around (1)} catalyzing an oxidative decolorization reaction    [decolorization activity] of each of:    Evans' Blue represented by the formula (I):

Acid Blue 80 represented by the formula (II):

Remazol Brilliant Blue R represented by the formula (III):

Acid Violet 17 represented by the formula (IV):

-   {circle around (2)} catalyzing oxidative degradation reaction    [oxidative degradation activity] for lignin;-   {circle around (3)} catalyzing oxidative polymerization reaction    [oxidative polymerization activity] of Indigo Carmine represented by    the formula (V):

Natural Orange 6 represented by the formula (VI):

-   {circle around (4)} catalyzing oxidative coupling reaction    [oxidative coupling activity] of 4-aminoantipyrine with one kind of    a compound selected from the group consisting of phenolic compounds,    aminophenolic compounds, diaminophenolic compound and heterocyclic    compounds; and-   {circle around (5)} catalyzing direct oxidative reaction [direct    oxidation activity] of one kind of a compound selected from the    group consisting of phenolic compounds, aminophenolic compounds,    diaminophenolic compounds and heterocyclic compounds;-   [2] the culture according to the above [1], wherein the strain    belonging to the genus Flammulina is Flammulina velutipes;-   [3] the culture according to the above [2], wherein Flammulina    velutipes is Flammulina velutipes strain IFO 30601;-   [4] the culture according to any one of claims 1 to 3, wherein the    culture is obtainable by culturing a strain belonging to the genus    Flammulina under pH conditions exceeding a pH of 7, and removing    hyphae from the resulting culture medium;-   [5] a culture from a strain belonging to the genus Flammulina,    wherein the culture is obtainable by culturing a strain belonging to    the genus Flammulina under pH conditions exceeding a pH of 7, and    removing hyphae from the resulting culture medium, wherein the    culture has phenol oxidase-like activity;-   [6] a method for producing the culture from a strain belonging to    the genus Flammulina according to any one of the above [1] to [5],    characterized in that the method comprises culturing a strain    belonging to the genus Flammulina, and removing hyphae from the    resulting culture medium, to give a culture;-   [7] the method according to the above [6], wherein the strain    belonging to the genus Flammulina is Flammulina velutipes;-   [8] the method according to the above [7], wherein Flammulina    velutipes is Flammulina velutipes IFO 30601 strain;-   [9] a staining method characterized in that the staining method    comprises contacting a subject to be stained with a dye in the    presence of the culture of any one of the above [1] to [5]; and-   [10] a staining composition comprising the culture of any one of the    above [1] to [5].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the results for examining the optimumtemperature of the culture of the present invention. Panel A of thefigure shows the case where 2,6-dimethoxyphenol was used as a substrate,and panel B shows the case where o-aminophenol was used as a substrate.

FIG. 2 is a diagram showing the results for examining the thermalstability of the culture of the present invention. Panel A of the figureshows the case where 2,6-dimethoxyphenol was used as a substrate, andpanel B shows the case where o-aminophenol was used as a substrate.

FIG. 3 is a diagram showing the results for examining the optimum pH ofthe culture of the present invention. Panel A of the figure shows thecase where 2,6-dimethoxyphenol was used as a substrate, panel B showsthe case where o-aminophenol was used as a substrate, and panel C showsthe case where p-phenylenediamine was used as a substrate.

FIG. 4 is a diagram showing the results for examining the pH stabilityof the culture of the present invention. Panels A and B show the casewhere 2,6-dimethoxyphenol is used as a substrate, and panels C and Dshow the case where o-aminophenol is used as a substrate. In addition,panels A and C show the pH stability after 1 hour, and panels B and Dshow the pH stability after 20 hours.

FIG. 5 is a diagram showing the results for staining the yak hair bundleand the wool cloth with the culture of the present invention. In FIG. 5,panel A shows the results using the yak hair bundle, and panel B showsthe results using the wool cloth.

FIG. 6 is a diagram showing the results for examining the change in theinduction of the activity depending upon the culture pH conditions.

FIG. 7 is a diagram showing the results comparing the optimum pH of thephenol oxidase-like activity of each of the culture of the presentinvention and the culture obtained by culturing at a pH of 6.0. Panels Aand B show optimum pH of the culture obtained by culturing at a pH of6.0, and panels C and D show optimum pH of the culture of the presentinvention. In addition, panels A and C show cases where2,6-dimethoxyphenol is used as a substrate, and panels B and D showcases where p-phenylenediamine is used as a substrate.

BEST MODE FOR CARRYING OUT THE INVENTION

The culture of the present invention has phenol oxidase-like activity,and can thus catalyze various chemical reactions resulting fromgeneration of radical species as reaction intermediates in the presenceof oxygen. In addition, the culture of the present invention possesses ahigh activity in the neutral range, with the optimum pH hardly changingdepending on the substrate used. Accordingly, the culture of the presentinvention exhibits excellent effects such as staining of fiber and hair,bleaching of pulp and fiber, removal of phenolic compounds from wasteliquor, degradation of endocrine disruptors, production of phenolicresin, production of artificial lacquer, production of an adhesive andthe like. Since the culture of the present invention is derived from astrain belonging to the genus Flammulina represented by edibleFlammulina velutipes described later, the culture of the presentinvention exhibits an excellent effect that the culture can be easilysupplied from culture products released outside of hyphae.

The strain belonging to the genus Flammulina includes, specificallyFlammulina velutipes, more specifically Flammulina velutipes strainIFO30601.

In the present specification, the phenol oxidase-like activity refers toan activity of catalytically oxidizing a phenolic compound, anaminophenolic compound, a phenylenediamine compound and the like in thepresence of oxygen. Such phenol oxidase-like activity can be measured,for example, by a coloring reaction due to a reaction of reducing anoxygen molecule with a phenol, aniline derivative, or the like as ahydrogen donor. The hydrogen donor includes a phenolic compound, anaminophenolic compound, a diaminophenolic compound, a heterocycliccompound, and the like.

The culture of the present invention has at least one substratespecificity selected from the group consisting of:

-   {circle around (1)} catalyzing oxidative decolorization reactions    [decolorization activity] of:    -   Evans' Blue represented by the formula (I):

-   -   Acid Blue 80 represented by the formula (II):

-   -   Remazol Brilliant Blue R represented by the formula (III):

-   -   Acid Violet 17 represented by the formula (IV):

-   {circle around (2)} catalyzing oxidative degradation reaction    [oxidative degradation activity] of lignin,-   {circle around (3)} catalyzing oxidative polymerization reaction    [oxidative polymerization activity]of Indigo Carmine represented by    the formula (V):

-   -   Natural Orange 6 represented by the formula (VI):

-   {circle around (4)} catalyzing oxidative coupling reaction    [oxidative coupling activity] of 4-aminoantipyrine with one kind of    a compound selected from the group consisting of a phenolic    compound, an aminophenolic compound, a diaminophenolic compound and    a heterocyclic compound, and-   {circle around (5)} catalyzing direct oxidative reaction [direct    oxidation activity] of one kind of a compound selected from the    group consisting of a phenolic compound, an aminophenolic compound,    a diaminophenolic compound and a heterocyclic compound.

The phenolic compound includes, for example, phenol, 2-methoxyphenol,2,6-dimethoxyphenol, catechol, resorcinol, hydroquinone, pyrogallol,gallic acid, propyl gallate, 1-naphthol, catechin and the like. Theaminophenolic compound includes, for example, o-aminophenol,m-aminophenol, p-aminophenol and the like. The diaminophenolic compoundincludes, for example, o-phenylenediamine, m-phenylenediamine,p-phenylenediamine and the like. The heterocyclic compound includes5-hydroxyindole, 2,6-diaminopyridine and the like.

Regarding the substrate specificity, the decolorization activity in theabove item {circle around (1)}, the oxidative degradation activity ofthe above item {circle around (2)}, and the oxidative polymerizationactivity of the above item {circle around (3)} were determined by:

-   a) a step of pre-incubating 950 μl of 105.3 mM sodium phosphate    buffer (pH 7.0) containing a substrate (dye, lignin or the like) at    25° C. for 5 minutes, to give a substrate solution,-   b) a step of mixing the substrate solution obtained in the step a)    with 50 μl of a culture solution containing the culture of the    present invention and then incubating the resulting mixture at    25° C. for 60 minutes, and-   c) a step of measuring the absorbance of the product obtained in the    step b), at a wavelength depending on the substrate. The    decolorization activity on the respective dyes can be evaluated by    measuring as the dye, Evans' Blue, Acid Blue 80, Remazol Brilliant    Blue R and Acid Violet 17 in the step c) for their absorbance at 600    nm, 600 nm, 600 nm and 550 nm respectively. In addition, regarding    the lignin, the oxidative degradation activity on the respective    dyes can be evaluated by measuring the absorbance at 450 nm in the    step c). The oxidative polymerization activity on the respective    dyes can be evaluated by measuring as the dye, Indigo Carmine and    Natural Orange 6 in the step c) for their absorbance at 600 nm and    450 nm respectively.

The oxidative coupling activity in the above item {circle around (4)} isdetermined by:

-   a) a step of pre-incubating 190 μl of 105.3 mM phosphate buffer (pH    7.0) containing 0.4 μmol hydrogen donor and 4.0 μmol    4-aminoantipyrine at 25° C. for 1 minute, to give a substrate    solution,-   b) a step of mixing the substrate solution obtained in the step a)    with 10 μl of a culture solution containing the culture of the    present invention, and then incubating the mixture at 25° C. for 60    minutes, and-   c) a step of measuring the absorbance of the product obtained in the    step b), at a wavelength depending on the hydrogen donor, wherein a    change in absorbance at 490 nm is measured when a phenolic compound,    a diaminophenolic compound or a heterocyclic compound is used as the    substrate, or a change in absorbance at 450 nm is measured when an    aminophenolic compound is used as the substrate.

The direct oxidation activity in the above item {circle around (5)} isdetermined by:

-   a) a step of pre-incubating 180 μl of 0.89 mM sodium phosphate    buffer (pH 7.0) containing 0.1 mmol substrate at 25° C. for 1    minute, to give a substrate solution,-   b) a step of mixing the substrate solution obtained in the step a)    with 20 μl of a culture solution containing the culture of the    present invention and then incubating the mixture at 25° C. for 60    minutes, and    -   c) a step of measuring a change in the absorbance of the product        obtained in the step b), at 490 nm, 450 nm or 405 nm depending        on the substrate.

The culture of the present invention exhibits an excellent phenoloxidase-like activity in a pH range of from 6.0 to 8.0. Since theculture of the present invention exhibits an excellent phenoloxidase-like activity on various phenolic compounds in the pH rangedescribed above, the culture exhibits an excellent property that it caneffect efficient reaction without particular pH adjustment.

The culture of the present invention exhibits an excellent phenoloxidase-like activity in the range of 20° to 60° C. Since the culture ofthe present invention exhibits an excellent phenol oxidase-like activityin the above temperature range, the culture exhibits a high activity inthe living temperature (room temperature, water temperature, bodytemperature, ambient temperature or the like) without regulating theconditions at specific temperature. Accordingly, staining, disposal ofwaste liquor, synthesis of polymer, and the like can be easily carriedout.

When the culture of the present invention is incubated at 30° C. for 1hour in a pH range of from 5.0 to 9.5, the culture of the presentinvention maintains a relative remaining activity of about 75% or moreas compared to the activity before incubation. In addition, when theculture of the present invention is incubated at 30° C. for 1 hour in apH range of from 4.0 to 10.5, the culture of the present inventionmaintains a relative remaining activity of about 40% or more as comparedto the activity before incubation. Regarding the pH stability of thephenol oxidase compound in the present invention, when the culture ofthe present invention is incubated at 30° C. for 20 hours, the culturemaintains a relative remaining activity of about 75% or more in a pHrange of from 7.0 to 9.0, as compared to the activity before incubation.Since the culture of the present invention exhibits excellent pHstability in the above pH range, the culture of the present inventionexhibits an excellent property that a water-based reaction solution canbe used without adjusting the conditions at specific pH.

In addition, when the culture of the present invention is incubated at apH of 6.0 for 1 hour at 0° to 40° C., the culture of the presentinvention maintains a relative remaining activity of about 75% or moreas compared to the activity before incubation. Since the culture of thepresent invention exhibits excellent thermal stability in the abovetemperature range, the culture of the present invention exhibits anexcellent property that it can be used and stored at ambienttemperatures without regulating the conditions at specific temperature.Accordingly, there can be exhibited an excellent effect that staining,disposal of waste liquor, synthesis of polymer, and the like can becarried out inexpensively and easily.

One of significant features of the culture of the present invention isalso resides in that the culture is a culture obtained by culturing astrain belonging to the genus Flammulina under pH conditions exceeding apH of 7, and then removing hyphae from the resulting culture solution.

The culture of the present invention is obtained by culturing a strainbelonging to the genus Flammulina under pH conditions exceeding a pH of7, and exhibits an excellent property that it shows a stable and highphenol oxidase-like activity in a pH range of from 6.0 to 8.0.

In the present specification, the “pH conditions exceeding a pH of 7”refer to conditions having a pH value higher than a pH exceeding a pH of7 and lower than a pH of 13.0, preferably a pH value of pHs of from 7.5to 11.0, more preferably a pH value of pHs of from 8.0 to 10.0.

The culture of the present invention may be a culture solution obtainedby culturing a strain belonging to the genus Flammulina, or may be aproduct obtained by removing hyphae from the culture solution.Therefore, the culture of the present invention can be provided easilyfrom a culture product outside of hyphae, obtained by culturing a strainbelonging to the genus Flammulina in a suitable medium. Since theremoved hyphae can be cultured in a new medium to give the culture ofthe present invention, the hyphae can be recycled. The present inventionencompasses a method for producing the culture of the present invention.

One of the significant features of the production method of the presentinvention resides in that the culture is obtained by culturing a strainbelonging to the genus Flammulina and then removing hyphae from theresulting culture medium.

According to the production method of the present invention, there canbe brought about an excellent effect that a culture having phenoloxidase-like activity can be obtained easily in a large amount byculturing a strain belonging to the genus Flammulina. Further, since theculture of the present invention can be obtained by culturing theremoved hyphae in a new medium to give the culture of the presentinvention, the removed hyphae can be recycled. Therefore, there isexhibited an excellent effect that the culture of the present inventioncan be obtained inexpensively and easily.

For culturing the strain belonging to the genus Flammulina, a liquid orsolid medium may be used.

The carbon source in the culture used in the production method of thepresent invention includes, as long as the carbon source may be a sourcewhich may be metabolized by the strain belonging to the genusFlammulina, sugar such as glucose, sucrose, molasses and starch; bran,mandarin orange pulp and the like. The carbon source can be used aloneor as a mixture of two or more thereof. The nitrogen source includes anorganic nitrogen source such as soybean fiber, peptone, trypton,casamino acid, yeast extract, malt extract, defatted soybean powder,corn steep liquor and urea; and an inorganic nitrogen source such aspotassium nitrate and ammonium sulfate. The nitrogen source can be usedalone or as a mixture of two or more thereof. The medium used in theproduction method of the present invention may be supplemented ifnecessary with inorganic salts such as phosphates, magnesium sulfate,magnesium carbonate, sodium carbonate, potassium, calcium, iron, copper,zinc, manganese and cobalt, vitamins and the like. The concentration ofthe carbon source, nitrogen source and the like in the medium may be aconcentration at which basidiomycetes producing the culture of thepresent invention, particularly a strain belonging to the genusFlammulina, can grow sufficiently to exhibit phenol oxidase-likeactivity, and specifically the carbon source is desired to be 0.1 to 20%by weight, preferably 1 to 10% by weight, and the nitrogen source isdesired to be 0.1 to 10% by weight, preferably 1 to 5% by weight.

The pH of the medium may be a pH at which a strain belonging to thegenus Flammulina can grow sufficiently to exhibit phenol oxidase-likeactivity, particularly phenol oxidase-like activity with the substratespecificity, further phenol oxidase-like activity showing the abovereaction optimum pH, and from the viewpoint of sufficiently exhibitingphenol oxidase-like activity with the above properties (for example,substrate specificity, reaction optimum pH and the like), the pH ispreferably exceeding a pH of 7.0 (initial pH), preferably pHs of from7.5 to 11.0, more preferably pHs of from 8.0 to 10.0. Accordingly, themedium is used desirably after adjustment to such pH and subsequentsterilization.

The culture temperature may be a temperature at which filamentous fungican grow, and is practically 10° to 40° C., preferably 20° to 35° C.

When the strain belonging to the genus Flammulina is cultured in aliquid medium, aeration culture or shake culture is desired.

The culture time is varied depending on various culture conditions, andin the case of aeration culture, it is desired that the culture time is2 to 10 days. The culture time can be set so as to allow the culturemedium to attain a highest activity.

The culture of the present invention is produced in secretion outside ofthe cell and accumulated in the culture solution. Accordingly, theculture solution can be used as it is, or can be obtained as aconditioned medium by removing unnecessary materials such as themicroorganism by centrifugation or filtration through a filter paper ora filter cloth after culture.

In the present invention, the phenol oxidase-like activity of theculture obtained as a culture solution or as a conditioned medium canfurther be increased or subjected to decolorization, concentration,freeze drying, dialysis, salting-out or the like from the viewpoint ofblending it into products such as cosmetics, and non-pharmaceuticalpreparations.

In addition, according to the culture of the present invention, stainingof keratin fibers can be carried out by using the culture together withvarious dyes, colorants and the like. Accordingly, the present inventionprovides a staining method.

One of the significant features of the staining method of the presentinvention resides in that a subject of staining is brought into contactwith a dye in the presence of the culture. According to the stainingmethod of the present invention, since the culture of the presentinvention is used, a subject of staining can be stained inexpensivelyand easily with various dyes. In addition, according to the stainingmethod of the present invention, since the culture of the presentinvention is used, the method can be carried out under externalenvironmental conditions without regulating conditions at specialtemperature or pH.

The subject of staining includes, for example, cotton, diacetate, flax,linen, liocel, polyacryl, polyamide, polyester, ramie, laen, tencel,triacetate, fur, animal skin, hide, silk or wool clothes, yarn, fiber,clothing, wood, hair, film and the like.

The dyes such as indoline and indoline compounds, indole compounds andoxidized dyes described in standards of materials of non-pharmaceuticalpreparations can be oxidized as substrates, and a coupling agent canalso be used.

Concretely, the indoline and the indoline compounds can be exemplifiedby indoline, 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline,N-ethyl-5,6-dihydroxyindoline, N-butyl-5,6-dihydroxyindoline,4-hydroxy-5-methoxyindoline, 6-hydroxy-7-methoxyindoline,6,7-dihydroxyindoline, 4,5-dihydroxyindoline,4-methoxy-6-hydroxyindoline, N-hexyl-5,6-dihydroxyindoline,2-methyl-5,6-dihydroxyindoline, 3-methyl-5,6-dihydroxyindoline,4-hydroxyindoline, 2,3-dimethyl-5,6-dihydroxyindoline,2-methyl-5-ethyl-6-hydroxyindoline,2-methyl-5-hydroxy-6-β-hydroxyethylindoline, 4-hydroxypropylindoline,2-hydroxy-3-methoxyindoline, 6-hydroxy-5-methoxyindoline,6-hydroxyindoline, 5-hydroxyindoline, 7-hydroxyindoline,7-aminoindoline, 5-aminoindoline, 4-aminoindoline,5,6-dihydroxyindolinecarboxylic acid, 1-methyl-5,6-dihydroxyindoline,and salts thereof, and the like.

Concretely, the indole compound includes 4-hydroxyindole,5-hydroxyindole, 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylicacid, 5,6-tri(t-butoxycarbonyloxy)indole,5,6-di(t-butoxycarbonyloxy)indole,5-t-butoxycarbonyloxy-6-hydroxyindole,6-t-butoxycarbonyloxy-5-hydroxyindole, 5,6-di(ethoxycarbonyloxy)indole,5,6-di(ethylcarbamoyloxy)indole,1-pivaloyl-5-(pivaloyloxymethoxy)-6-pivaloyloxyindole,1-pivaloyl-5-pivaloyloxymethoxy-6-hydroxyindole,5,6-(oxycarbonylmethoxy)indole, and the like.

Concretely, the oxidative dyes listed under the provision of rawmaterials of quasi-drugs can be exemplified by 5-amino-o-cresol,o-aminophenol, m-aminophenol, p-aminophenol, 2,6-diaminopyridine,5-(2-hydroxyethylamino)-2-methylphenol,N,N-bis(β-hydroxy)-p-phenylenediamine sulfate,p-nitro-o-phenylenediamine, sodium p-nitro-2′,4′-diaminoazobenzenesulfate, toluene-2,5-diamine, 5-amino-o-cresol sulfate, p-aminophenolsulfate, o-chloro-p-phenylenediamine sulfate, 4,4′-diaminodiphenylaminesulfate, p-methylaminophenol sulfate, p-phenylenediamine sulfate,m-phenylenediamine sulfate, toluene-2,5-diamine sulfate,2,4-diaminophenoxyethanol hydrochloride, toluene-2,5-diaminehydrochloride, m-phenylenediamine hydrochloride, 2,4-diaminophenolhydrochloride, 3,3′-iminodiphenol, p-phenylenediamine hydrochloride,N-phenyl-p-phenylenediamine hydrochloride, N-phenyl-p-phenylenediamineacetate, 1,5-dihydroxynaphthalene, toluene-3,4-diamine,p-methylaminophenol,N,N′-bis(4-aminophenyl)-2,5-diamino-1,4-quinonediimine, o-aminophenolsulfate, 2,4-diaminophenol sulfate, m-aminophenol sulfate, and the like.

In addition, in the present invention, there can be also used directdyes such as 2-amino-4-nitrophenol, 2-amino-5-nitrophenol,1-amino-4-methylaminoanthraquinone, nitro-p-phenylenediaminehydrochloride, 1,4-diaminoanthraquinone, nitro-p-phenylenediamine,picramic acid, sodium picramate, 2-amino-5-nitrophenol sulfate,resorcinol, nitro-p-phenylenediamine sulfate, p-nitro-o-phenylenediaminesulfate, p-nitro-m-phenylenediamine sulfate, and the like.

The contact of a subject of staining with a dye in the presence of theculture can be carried out, for example, by mixing the dye with theculture just before use and then contacting the mixture with the subjectof staining, or by contacting a mixture of the dye and the culturestored under anaerobic conditions, with the subject of staining in theair.

The culture of the present invention provides a staining composition.

One of significant features of the staining composition of the presentinvention resides in that the staining composition comprises the cultureof the present invention. Therefore, according to the stainingcomposition of the present invention, there is exhibited an excellenteffect that staining can be achieved efficiently with various dyes,particularly phenolic compounds, aminophenolic compounds, andphenylenediamine compounds, in the neutral pH range without significantchange in the optimum pH in the presence of oxygen and under externalenvironmental conditions.

From the viewpoint of practical staining time, the content of theculture of the present invention in the staining composition of thepresent invention is 0.05 to 100 KU, desirably 0.5 to 25 KU, as phenoloxidase-like activity, per 100 g of the hair staining composition. Oneunit (U) of phenol oxidase-like activity is defined as an amount forincreasing absorbance at 490 nm by 1 per minute with 30 mMp-phenylenediamine as substrate under the conditions of 25° C. and pH7.0.

In the staining composition of the present invention, there can be useddyes or colorants, specifically the above-mentioned indoline andindoline compounds, indole compounds, and oxidative dyes described instandards of materials of non-pharmaceutical preparations and the likecan be used. A coupling agent can also be used. The direct dyes can alsobe used.

From the viewpoint of practical staining time, it is desired that thecontent of the dye or colorant in the staining composition of thepresent invention is 0.0005 to 12% by weight, preferably 0.005 to 6% byweight, in the staining composition.

The pH of the staining composition of the present invention may beadjusted with an acid, alkali or buffer suitable for allowing theculture of the present invention to exhibit its physiological activity,thereby achieving the staining. Concretely, there are included inorganicacids such as hydrochloric acid, sulfuric acid etc.; organic acids suchas phosphoric acid, acetic acid, tartaric acid, citric acid, lacticacid, and sulfonic acid; ammonia; amines such as monoisopropanolamine,and monoethanolamine; carbonates such as ammonium and carbonate; andhydroxides such as sodium hydroxide, and potassium hydroxide.

In the staining composition, a reducing agent such as thiolactic acid,sodium sulfite, and N-acetyl-L-cysteine can be formulated thereto insuch a range that the phenol oxidase-like activity is not inhibited. Inaddition to the above-described components, a surfactant, an oilycomponent, silicone derivatives, a thickener, a solvent, water, achelating agent, a perfume and the like can be suitably formulated insuch a range that the effect of the present invention is not inhibited.

When the staining composition of the present invention is used as a hairstaining composition, a staining composition of one-pack type comprisinge.g. a dye and the culture is prepared under anaerobic conditions, andwhen used, the composition can be applied onto hair and contacted withthe air, to stain the hair. Alternatively, a composition comprising theculture, and a composition comprising a dye can be mixed with each otheras a staining composition of multi-pack type just before use and thenapplied onto hair, to stain the hair. The staining composition of thepresent invention can also be used in the form of various preparationssuitable for staining, such as liquid, cream and gel.

Since the culture of the present invention has phenol oxidase-likeactivity, the culture is useful not only in staining fiber and hair butalso in bleaching pulp and fiber, removing phenolic compounds in wasteliquor, degrading endocrine disruptors, producing phenolic resin,producing artificial lacquer, and improving woody properties.

The present invention will be described hereinbelow more specifically bymeans of Examples, without intending to limit the present invention tothese Examples.

EXAMPLE 1 Preparation of Culture of Flammulina velutipes (Strain IFO30601)

(1) Preparation 1 of Culture

The following procedures were carried out in a clean bench.

Flammulina velutipes (strain IFO 30601) was sown to a sterilized petridish containing 10 ml of a solid culture agar medium [composition: 2.4%by weight potato dextrose broth (manufactured by Difco), 2.0% by weightagar, and balance water] in an amount equivalent to a single loop ofplatinum, and cultured at 25° C. for 10 days. Thereafter, the hyphaegrown on the whole agar medium were cut into small pieces of 5 mm eachside with a sterilized loop of platinum.

Ten pieces of the above-mentioned small pieces were sown to a liquidculture medium 1 [composition: 2.4% by weight potato dextrose broth(manufactured by Difco), and balance water (pH 5.2); sterilized at 121°C. for 15 minutes], and culture with reciprocation shaking (150reciprocations/min.) was carried out at 25° C. for 7 days.

The entire volume of the resulting culture medium was added to 500 ml ofthe above-mentioned liquid culture medium in a 2-L Erlenmeyer flask, andculture with reciprocation shaking (100 reciprocations/min.) was carriedout at 25° C. for 3 weeks.

Thereafter, the grown pelletal hyphae were allowed to stand to beprecipitated, to remove the culture medium. In addition, 500 ml of aliquid culture medium 2 [composition: 1.0% by weight glucose, 0.1% byweight yeast extract, 0.14% by weight (NH₄)₂SO₄, 0.36% by weight K₂HPO₄,0.02% by weight MgSO₄.7H₂O, 0.1% mineral mixed solution (composition:1.0% by weight CuSO₄.5H₂O, 1.0% by weight ZnCl₂, 0.7% by weightFeCl₃.6H₂O, 0.5% by weight CoSO₄.7H₂O, 0.5% by weight MnCl₂.4H₂O), pH9.2; sterilized at 121° C. for 15 minutes] was added to the remaininghyphae, and culture was further carried out at 25° C. for 3 days.

Thereafter, the grown pelletal hyphae were allowed to stand to beprecipitated. The culture was collected by decantation. The resultingculture was a pale yellow or yellowish brown, transparent or turbidliquid.

(2) Preparation 2 of Culture

The culture of Flammulina velutipes strain IFO 30601 obtained in theabove (1) was filtered under reduced pressure, to give a filtrate.

The pH of the above-mentioned filtrate was adjusted to 7.5 with a 1 Maqueous sodium hydroxide. Five grams of DEAE-cellulose (manufactured bySigma) carrier was added to 1 L of the obtained mixture, and stirredwith shaking at 4° C. for 30 minutes. Thereafter, the mixture wasallowed to stand for 10 minutes, and supernatant was removed.

To the DEAE-cellulose carrier obtained was added 0.1 M sodium phosphatebuffer (pH 6.5) containing 1 M sodium chloride in an amount of 3 timesthe volume of the carrier. The mixture obtained was stirred with shakingfor 5 minutes to elute a protein adsorbed to the DEAE-cellulose carrier.The eluent obtained was collected by filtration under reduced pressureand dialyzed against deionized water at 4° C. The resulting product waslyophilized, to give a culture as a lyophilized product.

EXAMPLE 2 Reaction Optimum Temperature of Culture of the PresentInvention

One-hundred-and-two milligrams of the culture (lyophilized product)(amount of protein: 23 mg; specific activity: 15 U/mg protein) obtainedin (2) in Example 1 mentioned above was dissolved so as to have aconcentration of 0.02 mg protein/ml, to obtain a culture.

The phenol oxidase-like activity was assayed for the culture obtainedunder various temperature conditions (0°, 20°, 30°, 40°, 50°, 60°, 70°and 80° C.). Concretely, when o-aminophenol was used as a substrate,0.89 ml of 100 mM sodium phosphate buffer (pH 6.0), 0.1 ml of a dimethylsulfoxide solution of 50 mM o-aminophenol and 0.01 ml of the culturesolution were mixed in a microcentrifuge tube (manufactured by Porex BioProducts Incorporated), and the reaction was carried out for theresulting mixture at each reaction temperature for 10 minutes.Thereafter, 0.1 ml of 2 M glycine-HCl buffer (pH 3.0) was added to thereaction mixture, whereby stopping the reaction. Alternatively, when2,6-dimethoxyphenol was used as a substrate, the same procedures werecarried out as the case of o-aminophenol solution mentioned above exceptthat a 50 mM aqueous 2,6-dimethoxyphenol solution was used in place ofo-aminophenol.

Next, the activity was evaluated for each of the resulting reactionproducts by determining absorbance at 420 nm in the case whereo-aminophenol was used as a substrate, or determining absorbance at 470nm in the case where 2,6-dimethoxyphenol was used as a substrate, with aspectrophotometer (manufactured by Jasco, trade name: V-530). Here, theabove-mentioned activity by which absorbance was increased by 1 in oneminute was defined as 1 U (unit). The results are shown in FIG. 1. InFIG. 1, the activity is expressed as a relative activity, the maximumactivity of which is 100. In addition, panel A of the figure shows thecase where 2,6-dimethoxyphenol was used as a substrate, and panel Bshows the case where o-aminophenol was used as a substrate.

As shown in FIG. 1, it was seen that the reaction optimum temperature ofthe culture of the present invention is from 20° to 60° C.

EXAMPLE 3 Thermal Stability of Culture of the Present Invention

Pretreatment of 0.02 ml of the same culture solution as in Example 2mentioned above was carried out under various temperature conditions(0°, 20°, 30°, 40°, 50°, 60°, 70° and 80° C.) by incubating the culturesolution in 0.18 ml of 100 mM sodium phosphate buffer (pH 6.0) for 60minutes.

When o-aminophenol was used as a substrate, 0.05 ml of the resultingproduct, 0.85 ml of 100 mM sodium phosphate buffer (pH 6.0) and 0.1 mlof a dimethyl sulfoxide solution of 50 mM o-aminophenol weresufficiently mixed in a microcuvette. The activity was evaluated for theresulting mixture by determining a change in absorbance at 420 nm with aspectrophotometer (manufactured by Jasco, trade name: V-530).Alternatively, when 2,6-dimethoxyphenol was used as a substrate, thesame procedures were carried out as in the above-mentioned case whereo-aminophenol was used as a substrate by using 50 mM aqueous2,6-dimethoxyphenol solution in place of 50 mM o-aminophenol in thedimethyl sulfoxide solution, and thereafter, the activity was evaluatedby determining a change in absorbance at 470 nm. Here, 1 U (unit) of theactivity was defined as an amount for increasing the absorbance by 1 perone minute. The results are shown in FIG. 2. In FIG. 2, the activity isexpressed as a relative remaining activity, the maximum activity ofwhich is 100. In addition, panel A of the figure shows the case where2,6-dimethoxyphenol was used as a substrate, and panel B shows the casewhere o-aminophenol was used as a substrate.

As shown in FIG. 2, in the case of 2,6-dimethoxyphenol, the culture ofthe present invention showed a relative remaining activity of 84% at atemperature up to 40° C., a relative remaining activity of 45% at 50°C., and a relative remaining activity of 22% at 60° C. In the case ofo-aminophenol, the culture of the present invention showed a relativeremaining activity of 91% at a temperature up to 40° C., a relativeremaining activity of 62% at 50° C., and a relative remaining activityof 40% at 60° C.

EXAMPLE 4 Optimum pH of Culture of the Present Invention

During the reaction of the culture of the present invention with eachsubstrate, 0.2 M glycine-sodium hydroxide buffer (pH 11.5, pH 10.5, pH9.5), 0.2 M Tris-HCl buffer (pH 10.0, pH 9.0, pH 8.0, pH 7.0), 0.2 Msodium phosphate buffer (pH 7.5, pH 7.0, pH 6.5, pH 6.0, pH 5.5), 0.2 Msodium acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5) and 0.2 Mglycine-HCl buffer (pH 4.0, pH 3.5, pH 3.0, pH 2.0) were used as buffersdepending upon the pHs.

When o-aminophenol was used as a substrate, 0.02 ml of the same culturesolution as in Example 2 mentioned above, 0.88 ml of one of theabove-mentioned various buffers, 0.1 ml of a dimethyl sulfoxide solutionof 50 mM o-aminophenol were sufficiently mixed in a microcuvette. Theactivity was evaluated for the resulting mixture by determining a changein absorbance at 420 nm with a spectrophotometer (manufactured by Jasco,trade name: V-530). Alternatively, when 2,6-dimethoxyphenol was used asa substrate, the same procedures were carried out as in theabove-mentioned case where o-aminophenol was used as a substrate byusing 50 mM aqueous 2,6-dimethoxyphenol solution in place of thedimethyl sulfoxide solution of 50 mM o-aminophenol, and thereafter, theactivity was evaluated by determining a change in absorbance at 470 nm.

When p-phenylenediamine was used as a substrate, 0.1 M Tris-HCl buffer(pH 9.0, pH 8.5, pH 8.0, pH 7.5, pH 7.0), 0.1 M sodium phosphate buffer(pH 9.0, 8.5, 8.0, pH 7.5, pH 7.0, pH 6.5, pH 6.0) and 0.1 M sodiumcitrate buffer (pH 8.0, pH 7.5, pH 7.0, pH 6.5, pH 6.0, pH 5.0, pH 4.0,pH 3.0) were used as buffers depending upon the pHs. The activity wasevaluated by mixing sufficiently 5 μl of the above-mentioned culturesolution, 0.895 ml of the above-mentioned various buffers and 100 mMaqueous p-phenylenediamine in a microcuvette, and then determining forthe resulting mixture a change in absorbance at 490 nm with aspectrophotometer (manufactured by Jasco, trade name: V-530).

Here, 1 U (unit) of the activity was defined as an amount for increasingthe absorbance by 1 per one minute. The results are shown in FIG. 3. InFIG. 3, the activity is expressed as a relative activity, the maximumactivity of which is 100. In addition, panel A of the figure shows thecase where 2,6-dimethoxyphenol was used as a substrate, panel B showsthe case where o-aminophenol was used as a substrate, and panel C showsthe case where p-phenylenediamine was used as a substrate.

As shown in FIG. 3, it was found that the optimum pH of the culture ofthe present invention is about 6.0 when 2,6-dimethoxyphenol is used as asubstrate, that the optimum pH is about 6.0 when o-aminophenol is usedas a substrate, and that the optimum pH is 6.5 to 8.0 whenp-phenylenediamine is used as a substrate.

EXAMPLE 5 pH Stability of Culture of the Present Invention

Pretreatment was carried out by mixing 0.02 ml of the same culturesolution as in Example 2 mentioned above with 0.18 ml of one of thevarious buffers having pHs of from 2 to 11.5, and then incubating themixture at 30° C. for 1 hour or 20 hours. During the pretreatment, 0.2 Mglycine-sodium hydroxide buffer (pH 11.5, pH 10.5, pH 9.5), 0.2 MTris-HCl buffer (pH 9.0, pH 8.0), 0.2 M sodium phosphate buffer (pH 7.0,pH 6.0), 0.2 M sodium acetate buffer (pH 5.0, pH 4.0) and 0.2 Mglycine-HCl buffer (pH 3.0, pH 2.0) were used as buffers depending uponthe pHs.

Next, the activity was evaluated by mixing sufficiently 0.02 ml of theculture solution after the pretreatment, 0.88 ml of the above-mentionedbuffer, and 0.1 ml of 50 mM o-aminophenol in dimethyl sulfoxide solutionin a microcuvette and then determining for the resulting mixture achange in absorbance at 420 nm with a spectrophotometer (manufactured byJasco, trade name: V-530). Alternatively, when 2,6-dimethoxyphenol wasused as a substrate, the same procedures were carried out as in theabove-mentioned case where o-aminophenol was used as a substrate byusing a 50 mM aqueous 2,6-dimethoxyphenol solution in place of thedimethyl sulfoxide solution of 50 mM o-aminophenol. The activity wasevaluated by determining a change in absorbance at 470 nm. Here, 1 U(unit) of the activity was defined as an amount for increasing theabsorbance by 1 per one minute. The results are shown in FIG. 4. In FIG.4, the activity is expressed as a relative remaining activity, themaximum activity of which is 100. In addition, panels A and B show thecase where 2,6-dimethoxyphenol is used as a substrate, and panels C andD show the case where o-aminophenol is used as a substrate. Further,panels A and C show the pH stability after 1 hour, and panels B and Dshow the pH stability after 20 hours.

As shown in FIG. 4, in the case of the 1-hour treatment, the culture ofthe present invention is stable at pHs of from 6.0 to 10.0 and retains arelative remaining activity of 75%. Also, in the case of the 20-hourtreatment, the culture is stable within a pH range of from 7.0 to 9.0and retains a relative remaining activity of 75%.

EXAMPLE 6 Substrate Specificity of Culture of the Present Invention

In order to examine the substrate specificity of the culture medium, thesubstrate specificity was examined by using p-phenylenediamine,4-hydroxyindole, 2-methoxyphenol, 2,6-dimethoxyphenol, catechol,p-aminophenol, o-aminophenol, ABTS, syringaldazine and L-tyrosine wereused as substrates. Concretely, the activity was evaluated by mixingsufficiently 0.05 ml of the above-mentioned culture solution, 0.85 ml of0.1 M sodium phosphate buffer (pH 7.0) with 0.1 ml of a substratesolution (L-tyrosine was used in a concentration of 1.1 mM,syringaldazine was used in a concentration of 1 mM, and others were usedin a concentration of 50 mM) in a microcuvette, and determining for theresulting mixture a change in absorbance at 420 nm with aspectrophotometer (manufactured by Jasco, trade name: V-530). Here, 1 U(unit) of the activity was defined as an amount for increasing theabsorbance by 1 per one minute. The results are shown in Table 1.

TABLE 1 Determined Wavelength Substrate (nm) U p-Phenylenediamine 4700.101 4-Hydroxyindole 470 0.078 2-Methoxyphenol 470 0.1172,6-Dimethoxyphenol 470 0.830 Catechol 400 0.029 p-Aminophenol 400 0.072o-Aminophenol 420 0.262 ABTS 420 0.683 Syringaldazine 530 0.141L-Tyrosine 490 0.018

From the results of Table 1, it was found that the culture of thepresent invention has phenol oxidase-like activity.

Next, chemical changes of various substrates generated by the action ofthe culture for the substrate specificity of the culture obtained in (2)of Example 1 was examined by determining a change in the absorbance ofthe buffer containing each substrate.

As the substrate, commercially available pigments (Evans' Blue, AcidBlue 80, Remazol Brilliant Blue R, Indigo Carmine, Acid Violet 17,Natural Orange) and lignin (trade name: Lignin Alkali, manufactured byTokyo Kasei) were used. Here, the final concentration and the measuringwavelength for each substrate are shown in Table 2.

TABLE 2 Final Concen- Measuring tration Wavelength Substrate (g/ml) (nm)Azo-Based Pigment Evans' Blue 0.007 600 Anthraquinone- Acid Blue 80 0.03600 Based Pigment Remazol Brilliant Blue R 0.23 600 Other PigmentsIndigo Carmine 0.005 600 Acid Violet 17 0.01 550 Natural Orange 6 0.03450 Lignin 1.00 450

Concretely, 950 μl of 105.3 mM sodium phosphate buffer (pH 7.0)containing a substrate adjusted to have a final concentration as shownin Table 2 was pre-incubated at 25° C. for 5 minutes. Next, 50 μl of theculture solution was added to the resulting product, and thereafter, themixture was reacted in a cuvette. The absorbance for each substrate wasdetermined at a measuring wavelength with a spectrophotometer(manufactured by Shimadzu Corporation, trade name: UV-2450). For thecomparison, the examination was also made for bilirubin oxidase from astrain belonging to the genus Myrothecium in the same manner byreplacing the above-mentioned buffer with 105.3 mM Tris-HCl buffer (pH8.0).

Here, 1 U (unit) of the activity was defined as an amount for increasingthe absorbance by 1 per one minute. As to the activity of the culture,the specific activity of each substrate (U/mg protein) is shown in Table3. In Table 3, a specific activity shown without a sign indicatesdegradation activity or decolorization activity, and a specific activityshown with—(minus) indicates polymerization activity.

TABLE 3 Specific Activity (U/mg Protein) Bilirubin Oxidase from StrainBelonging to Measuring Culture Genus Myrothecium Wavelength Reaction pHSubstrate (nm) 7.0 8.0 Azo-Based Pigment Evans' Blue 600 0.32 1.22Anthraquinone-Based Pigment Acid Blue 80 600 0.30 0.39 Remazol Brilliant600 0.97 −0.18 Blue R Other Pigments Indigo Carmine 600 −0.22 0.46 AcidViolet 17 550 0.12 −0.40 Natural Orange 6 450 −0.18 −0.11 Lignin 4500.73 −1.63

As shown in Table 3, it was found that under neutral conditions, theculture of the present invention catalyzed the oxidative decolorizationreaction of the azo-based pigment, the anthraquinone-based pigments andAcid Violet 17; the oxidative degradation reaction of lignin; and theoxidative polymerization reaction of Indigo Carmine and Natural Orange6. In particular, the culture strongly catalyzed the oxidativedecolorization reaction of Remazol Brilliant Blue R and the oxidativedegradation reaction of lignin. In addition, the culture of the presentinvention significantly differs in the specificities for RemazolBrilliant Blue R, Indigo Carmine, Acid Violet 17 and lignin from thoseof the bilirubin oxidase of a strain belonging to the genus Myrotheciumused for comparison.

Next, the oxidative coupling reaction of the culture obtained in (2) inExample 1 mentioned above was determined by using color development asan index by the oxidative condensation of the substrate (hydrogen donor)with 4-aminoantipyrine, catalyzed by the culture of the presentinvention.

As the hydrogen donors, there were used phenolic compounds (phenol,2-methoxyphenol, 2,6-dimethoxyphenol, catechol, resorcinol,hydroquinone, pyrogallol, gallic acid, propyl gallate, 1-naphthol,catechin), aminophenolic compounds (o-aminophenol, m-aminophenol,p-aminophenol), diaminophenolic compounds (o-phenylenediamine,m-phenylenediamine, p-phenylenediamine), and heterocyclic compounds(5-hydroxyindole, 2,6-diaminopyridine).

The above-mentioned culture solution was added to a reaction systemcontaining 4-aminoantipyrine and an optional hydrogen donor. Thereafter,the activity of the culture of the present invention was assayed bydetermining a change in absorbance at 490 nm for the phenolic compound,the diaminophenolic compound and the heterocyclic compound, ordetermining a change in absorbance at 450 nm for the aminophenoliccompound. As to a substrate hardly soluble in water, there was used oneobtained by dissolving the substrate in a small amount of dimethylsulfoxide and diluting the resulting solution with deionized water to adesired concentration. The measuring wavelength and the finalconcentration of each substrate are shown in Table 4. In Table 4, DMSOis an abbreviation for dimethyl sulfoxide.

TABLE 4 Measuring Final Wavelength Concentration Substrate (nm) (mM)Phenolic Compounds Phenol 490 2.0 2-Methoxyphenol 490 2.02,6-Dimethoxyphenol 490 2.0 Catechol 490 2.0 Resorcinol 490 2.0Hydroquinone 490 2.0 Pyrogallol 490 2.0 Gallic acid 490 2.0 PropylGallate 490 2.0 (DMSO 1%) 1-Naphthol 490 2.0 (DMSO 1%) Catechin 490 2.0(DMSO 1%) Aminophenolic Compounds o-Aminophenol 450 2.0 (DMSO 1%)m-Aminophenol 450 2.0 (DMSO 1%) p-Aminophenol 450 2.0 (DMSO 1%)Diaminophenolic Compounds o-Phenylenediamine 490 2.0 m-Phenylenediamine490 2.0 p-Phenylenediamine 490 2.0 Heterocyclic Compounds5-Hydroxyindole 490 2.0 (DMSO 1%) 2,6-Diaminopyridine 490 2.0 (DMSO 1%)In the table, “%” indicates volume/volume %.

Concretely, 190 μl of 105.3 mM phosphate buffer (pH 7.0) containing 0.4μmol of a substrate and 4.0 μmol of 4-aminoantipyrine was pre-incubatedin wells of a 96-well microplate [manufactured by Corning, Costor(registered trademark) 3368] at 25° C. for 1 minute. Next, 10 μl of theabove-mentioned culture solution was added to the resulting mixture, andthereafter, the mixture was incubated for 1 hour. A change in absorbanceat 490 nm or 450 nm was determined for the resulting product. One unit(U) of the activity regarding the oxidative coupling reaction wasdefined as an amount for increasing the absorbance by 1 per one minute.

In addition, for the comparison, the examination was made in the samemanner for bilirubin oxidase from the strain of the genus Myrotheciumexcept that the above-mentioned buffer was replaced with 105.3 mMTris-HCl buffer (pH 8.0). The specific activity (U/mg protein) for eachsubstrate is shown in Table 5.

TABLE 5 Specific Activity (U/mg Protein) Bilirubin Oxidase from StrainBelonging to Culture Genus Myrothecium Reaction pH Substrate 7.0 8.0Phenolic Compounds Phenol 8.1 0.9 2-Methoxyphenol 14.5 2.52,6-dimethoxyphenol 18.1 12.0 Catechol 23.3 16.0 Resorcinol 5.7 25.6Hydroquinone 2.1 0.5 Pyrogallol 3.2 −27.6 Gallic acid 0.9 14.9 PropylGallate −0.2 −0.1 1-Naphthol 18.1 33.0 Catechin 14.0 4.6 AminophenolicCompounds o-Aminophenol 22.4 15.1 m-Aminophenol 8.8 1.3 p-Aminophenol12.7 7.0 Diaminophenolic Compounds o-Phenylenediamine 15.1 4.5m-Phenylenediamine 29.7 1.7 p-Phenylenediamine 20.9 11.1 HeterocyclicCompounds 5-Hydroxyindole 4.7 1.9 2,6-Diaminopyridine 37.7 2.3

As shown in Table 5, the culture of the present invention catalyzed theoxidative coupling reaction of various phenolic compounds, aminophenoliccompounds and diaminophenolic compounds, with the aniline compound4-aminoantipyrine under neutral conditions (pH 7.0). As to the phenoliccompounds, the culture of the present invention was found to have a highactivity for the compounds other than gallic acid and propyl gallate. Inparticular, the culture was found to have an especially high activityfor 2-methoxyphenol, 2,6-dimethoxyphenol, catechol, 1-naphthol andcatechin. As to the aminophenolic compounds, the culture was found tohave a high activity for all of the compounds. In particular, theculture was found to have an especially high activity for o-aminophenoland p-aminophenol. As to the diaminophenolic compounds, the culture wasfound to have a high activity for all of the compounds. As to theheterocyclic compounds, the culture was found to have a high activityfor all of the compounds. In particular, the culture was found to have ahigh activity for 2,6-diaminopyridine.

In addition, as shown in Table 5, it was found that the culture of thepresent invention has a substrate specificity significantly differentfrom that of the bilirubin oxidase from the strain belonging to thegenus Myrothecium, and exhibits a higher specific activity.

Further, the direct oxidation reaction of the culture obtained in theabove-mentioned example was determined by using color development due tooxidative polymerization of a substrate generated by the direct actionof the culture of the present invention on the substrate as an index.

As the substrates, there were used diaminophenolic compounds (2-chloro1,4-phenylenediamine, p-phenylenediamine, 2,5-diaminotoluene,N-phenyl-p-phenylenediamine, toluylene-3,4-diamine, m-phenylenediamine),aminophenolic compounds (o-aminophenol, m-aminophenol, 5-amino-o-cresol,p-aminophenol, p-methylaminophenol), and phenolic compounds(2,6-dimethoxyphenol, catechol, resorcinol, hydroquinone, pyrogallol,gallic acid, propyl gallate, 1-naphthol, 1,5-dihydronaphthalene,2,3,4-trihydroxybenzophenone, 2,3,4,4-tetrahydroxybenzophenone). As to asubstrate hardly soluble in water, there was used one obtained bydissolving the substrate in a small amount of dimethyl sulfoxide anddiluting the resulting solution with deionized distilled water to adesired concentration. The measuring wavelength and the finalconcentration of each substrate are shown in Table 6. In Table 6, %”indicates volume/volume %.

TABLE 6 Measuring Final Wavelength Concentration Substrate (nm) (mM)Diaminophenolic Compounds 2-Chloro 1,4-phenylenediamine 490 0.50 mM(DMSO 1%) p-Phenylenediamine 490 0.50 mM (DMSO 1%) 2,5-Diaminotoluene490 0.50 mM (DMSO 1%) N-Phenyl-p-phenylenediamine 490 0.50 mM (DMSO 1%)Toluylene-3,4-diamine 490 0.25 mM (DMSO 1%) m-Phenylenediamine 405 0.50mM (DMSO 1%) Aminophenolic Compounds o-Aminophenol 450 0.50 mM (DMSO 1%)m-Aminophenol 405 0.50 mM (DMSO 1%) 5-Amino-o-cresol 450 0.50 mM (DMSO1%) p-Aminophenol 450 0.50 mM (DMSO 1%) p-Methylaminophenol 450 0.50 mM(DMSO 1%) Phenolic Compounds 2,6-Dimethoxyphenol 450 0.50 mM (DMSO 1%)Catechol 405 0.50 mM (DMSO 1%) Resorcinol 405 0.50 mM (DMSO 1%)Hydroquinone 405 0.50 mM (DMSO 1%) Pyrogallol 405 0.50 mM (DMSO 1%)Gallic Acid 405 0.50 mM (DMSO 1%) Propyl Gallate 405 0.50 mM (DMSO 1%)1-Naphthol 405 0.50 mM (DMSO 1%) 1,5-Dihydronaphthalene 405 0.50 mM(DMSO 1%) 2,3,4-Trihydroxybenzophenone 450 0.25 mM (DMSO 1%)2,3,4,4-Tetrahydroxybenzophenone 450 0.25 mM (DMSO 1%)

Concretely, 180 μl of 0.89 mM sodium phosphate buffer (pH 7.0)containing 0.1 mmol substrate was pre-incubated in wells of a 96-wellmicroplate [manufactured by Corning, Costor (registered trademark) 3368]at 25° C. for 1 minute. Next, 20 μl of the above-mentioned culturesolution was added to the resulting mixture. A change in absorbance at490 nm, 450 nm or 405 nm was determined for the resulting product,depending on the substrate. Here, 1 U (unit) of the activity regardingthe direct oxidative reaction was defined as an amount for increasingthe absorbance by 1 per one minute.

In addition, for the comparison, the substrate specificities at pH 7.0for laccase from Japanese lacquer and bilirubin oxidase from the strainbelonging to the genus Myrothecium were also examined in the samemanner.

The relative activity (%) for each substrate is shown in Table 7, inwhich the measured value for a case where a representative chemical dye2-chloro 1,4-phenylenediamine was used as a substrate is 100.

TABLE 7 Relative Activity (%) Laccase Bilirubin Oxidase from from StrainJapanese Belonging to Culture Lacquer Genus Myrothecium Reaction pHSubstrate 7.0 7.0 7.0 Diaminophenolic Compounds 2-Chloro 1,4- 100 100100 phenylenediamine p-Phenylenediamine 388 142 82 2,5-Diaminotoluene 8044 24 N-Phenyl-p- 174 94 102 phenylenediamine Toluylene-3,4-diamine 12061 45 m-Phenylenediamine 26 4 14 Aminophenolic Compounds o-Aminophenol650 103 86 m-Aminophenol 40 3 3 5-Amino-o-cresol 117 1 19 p-Aminophenol235 109 70 p-Methylaminophenol 304 308 122 Phenolic Compounds2,6-Dimethoxyphenol 250 21 69 Catechol 21 2 5 Resorcinol 75 25 91Hydroquinone 20 2 4 Pyrogallol 184 187 104 Gallic Acid 65 9 88 PropylGallate 34 14 85 1-Naphthol 17 17 50 1,5-Dihydronaphthalene 63 47 692,3,4-Trihydroxy- 537 175 62 benzophenone 2,3,4,4-Tetrahydroxy- 571 17952 benzophenone

As shown in Table 7, the culture of the present invention catalyzed thedirect oxidation reaction of various diaminophenolic compounds,aminophenolic compounds and phenolic compounds under neutral conditions(pH 7.0). As to the diaminophenolic compounds, the culture was found tohave a high activity for the compounds other than m-phenylenediamine. Inparticular, the culture was found to have an especially high activityfor p-phenylenediamine and N-phenyl-p-phenylenediamine. As to theaminophenolic compounds, the culture was found to have a high activityfor the compounds other than m-aminophenol. In particular, the culturewas found to have an especially high activity for o-aminophenol,p-aminophenol and p-methylaminophenol. As to the phenolic compounds, theculture was found to have a high activity for 2,6-dimethoxyphenol,pyrogallol, 2,3,4-trihydroxybenzophenone, and2,3,4,4-tetrahydroxybenzophenone. In particular, the culture was foundto have an especially high activity for 2,3,4-trihydroxybenzophenone and2,3,4,4-tetrahydrobenzophenone.

Also, the culture of the present invention has a higher activity forp-phenylenediamine, m-phenylenediamine, o-aminophenol, m-aminophenol,5-amino-o-cresol, p-aminophenol, 2,6-dimethoxyphenol, catechol,resorcinol, hydroquinone, gallic acid, propyl gallate,2,3,4-trihydroxybenzophenone and 2,3,4,4-tetrahydroxybenzophenone, thanthat of the laccase from Japanese lacquer. In particular, the culturewas found to have an even higher activity for m-phenylenediamine,o-aminophenol, m-aminophenol, 5-amino-o-cresol, 2,6-dimethoxyphenol,catechol, hydroquinone and gallic acid.

In addition, the culture of the present invention has a higher activityfor p-phenylenediamine, 2,5-diaminotoluene, toluylene-3,4-diamine,o-aminophenol, m-aminophenol, 5-amino-o-cresol, p-aminophenol,p-methylaminophenol, 2,6-dimethoxyphenol, catechol, hydroquinone, propylgallate, 1-naphthol, 2,3,4-trihydroxybenzophenone and2,3,4,4-tetrahydroxybenzophenone, than that of the bilirubin oxidasefrom the strain belonging to the genus Myrothecium. In particular, theculture was found to have an even higher activity for o-aminophenol,m-aminophenol, 5-amino-o-cresol, 2,6-dimethoxyphenol, hydroquinone,1-naphthol, 2,3,4-trihydroxybenzophenone and2,3,4,4-tetrahydroxybenzophenone.

EXAMPLE 7 Staining with Culture of the Present Invention

(1) Stainability with Culture of the Present Invention

A staining test was carried out with the culture obtained in Example 1mentioned above. A yak hair bundle and a wool cloth were stained withdyes (p-phenylenediamine and o-aminophenol) with the above-mentionedculture by oxidative polymerization. Concretely, 0.5 g of a dye (or 1.0g in total when the 2 kinds of dyes were used), 0.75 g of hydroxyethylcellulose (HEC) as a thickener, 1.0 g of polyoxyethylene(20)hydrogenated castor oil (HC-20) as a surfactant, and 0.5 g of lacticacid were mixed, and the pH was adjusted to 7.0 with monoethanolamine.Deionized water was added to the mixture to make up a weight of 50 g, togive a base material.

Two grams of the above-mentioned base material and the above-mentionedculture solution (amount equivalent to 3.5 U) were mixed, and themixture obtained was applied to each of 1 g of yak hair bundle and onepiece of a wool cloth (2×3 cm). Each of the yak hair bundle and the woolcloth after the application was kept at 30° C. for 1 hour. The activityin the above-mentioned culture solution is an activity determined using30 mM p-phenylenediamine as the substrate under the conditions of pH 7.0and 25° C.

L value, a value and b value for the stained yak hair bundle and thewool cloth were determined with a colorimeter (manufactured by Minolta,trade name: Chromometer CM-3610d). Next, on the bases of theabove-mentioned L value, a value and b value, ΔE value was calculatedfrom the equation 1:

${\Delta\; E} = \sqrt{\left( {\; L} \right)^{2} + \left( {\; a} \right)^{2} + \left( {\; b} \right)^{2}}$The stainability of the culture was evaluated from the above ΔE value.The ΔE value indicates a difference between the color tone of the yakhair bundle and the wool cloth before staining and the color tone of theyak hair bundle and the wool cloth after staining. For the comparison, astaining test was carried out in the same manner as the laccase fromJapanese lacquer. The ΔE value for each substrate is shown in Table 8.

TABLE 8 ΔE Yak Hair Bundle Wool Cloth p-Phenylenediamine Without Enzyme 9.39  8.02 Laccase from 43.66 49.58 Japanese Lacquer (34.27) (41.56)Culture 42.22 44.53 (32.83) (36.51) o-Aminophenol Without Enzyme 16.8414.74 Laccase from 43.91 38.27 Japanese Lacquer (27.07) (23.53) Culture45.63 48.20 (28.79) (33.46) o-Aminophenol + Without Enzyme 20.23 14.01p-Phenylenediamine Laccase from 33.26 41.35 Japanese Lacquer (13.03)(27.34) Culture 33.23 39.17 (13.00) (25.16) o-Aminophenol + WithoutEnzyme 20.52 10.59 m-Phenylenediamine Laccase from 32.66 26.30 JapaneseLacquer (12.14) (15.71) Culture 38.38 45.54 (17.86) (34.95)

As shown in Table 8, according to the culture of the present invention,evident stainability was recognized for the yak hair bundle and the woolcloth in any combinations of dyes. Also, according to the culture of thepresent invention, it was found that although the stainability withp-phenylenediamine alone or with o-aminophenol+p-phenylenediamine wasslightly inferior, the stainability with o-aminophenol alone and witho-aminophenol+m-phenylenediamine was evidently superior, than that ofthe laccase from Japanese lacquer used for comparison.

(2) Studies on Amount Used of Culture of the Present Invention

A staining test was carried out with the culture of the presentinvention with varying amounts of the culture obtained in Example 1mentioned above. Using a dye (o-aminophenol) which is to be oxidizedwith the culture of the present invention, the yak hair bundle and thewool cloth were stained by oxidative polymerization, and L value, avalue and b value were determined by a calorimeter.

Concretely, 0.5 g of o-aminophenol as a dye (substrate), 0.5 g ofm-phenylenediamine as a coupler, 0.75 g of hydroxyethyl cellulose (HEC)as a thickener, 1.0 g of polyoxyethylene(20) castor oil (HC-20) as asurfactant, and 0.5 g of lactic acid were mixed, and the pH of themixture was adjusted to 7.0 with monoethanolamine. Deionized water wasadded to the mixture to make up a weight of 50 g, to give a basematerial.

In addition, for 1 g of the yak hair bundle and 1 piece of the woolcloth (2×3 cm), 2 g of the above-mentioned base material and a culturesolution adjusted so as to contain 0 to 0.8 mg as an amount of proteinin the culture. The resulting mixture was applied to 1 g of the yak hairbundle and 1 piece of the wool cloth (2×3 cm). Each of the yak hairbundle and the wool cloth after the application was kept at 30° C. for 1hour.

L value, a value and b value were determined for the stained yak hairbundle and the wool cloth with a colorimeter (manufactured by Minolta,trade name: Chromometer CM-3610d), and ΔE value was calculatedtherefrom. The stainability of the culture was evaluated by the ΔEvalue. A change in ΔE value according to the amount of the culture ofthe present invention used is shown in FIG. 5. Panel A of FIG. 5 showsthe results using the yak hair bundle, and panel B shows the resultsusing the wool cloth.

As shown in FIG. 5, in the yak hair bundle and the wool cloth, the ΔEvalue increased by increasing the amount of the culture of the presentinvention used. The ΔE value in the yak hair bundle reached a givenvalue by the culture of the present invention in an amount equivalent to0.2 mg protein, while the ΔE value in the wool cloth reached a givenvalue by the culture of the present invention in an amount equivalent to0.5 mg protein.

EXAMPLE 8 Studies on Conditions for Inducing Phenol Oxidase-LikeActivity

(1) Culture of Fungal Cells

The culture (liquid culture) obtained in (1) in Example 1 mentionedabove was cultured in the liquid culture medium 2 (medium described inExample 1) under five culture pH conditions within the range of weaklyacidic to weakly alkaline (initial pH: pH 5.0, pH 6.0, pH 7.0, pH 8.0,pH 9.0), and a difference in the oxidative activity (induction of phenoloxidase-like activity) was examined.

The above-mentioned liquid culture was stirred well, and thereafter, 0.5ml of the resulting culture was added to 100 ml of a medium for inducingthe activity. The resulting mixture was cultured with shaking (shakingrate 130 rotations/minute) in a thermostatic bath at 28° C. for 10 days.

The culture was collected 1 mg every day and centrifuged at 12000 rpm at4° C. for 5 minutes. Supernatant was filtered with DISMIC-25cs (0.20 μm)to remove the Fungal Cells.

The sample obtained was preserved at 4° C.

(2) Studies on Change in Induction of Activity According to Culture pHConditions

The change in induction of the activity according to culture pHconditions was examined by assaying the phenol oxidase-like activity ofeach sample obtained in the above-mentioned (1).

In the assay of the phenol oxidase-like activity, the phenoloxidase-like activity was assayed under the reaction pH condition of pH6.0 by using 2,6-dimethoxyphenol as a substrate.

To a semimicrocuvette were added 0.20 ml of 500 mM sodium phosphatebuffer (pH 6.0), 0.65 ml of deionized distilled water, 0.10 ml of 50 mMsubstrate solution mentioned above and 0.05 ml of the culture, withstirring. The change in absorbance at 470 nm from 0 to 30 seconds atroom temperature was determined with trade name: UV-2400 (manufacturedby Shimadzu Corporation). The change in induction of the activityaccording to the culture pH conditions is shown in FIG. 6.

As a result, as shown in FIG. 6, it is found that the higher the pH ofthe medium used is, the higher the enzyme activity at a pH of 6.0, whichis the reaction optimum pH for 2,6-dimethoxyphenol. Especially, when thepH of the medium used is a value exceeding a pH of 7.0 (pH of about 7.3during the culture), it is found that the enzyme activity at a pH of6.0, which is the reaction optimum pH for 2,6-dimethoxyphenol, becomeshigh.

On the other hand, when the pH of the medium used is a pH of 5.0, theenzyme activity for 2,6-dimethoxyphenol was not recognized even when thebacterial cells were cultured for 10 days.

In addition, as shown in FIG. 6, when the pH of the medium used is a pHof 9.0, it is seen that the activity on the tenth day of the culture isabout 10 times (0.283/0.027) higher than the activity on the tenth dayof the culture when the pH of the medium used is a pH of 6.0.

Therefore, it is found that the phenol oxidase-like activity in theculture is more induced by using the medium having a pH value of higherthan 7.0.

TEST EXAMPLE

(1) Culture of Fungal Cells

Two-hundred-and-fifty grams of soybeans were disrupted with a mixer, thedisruption obtained was placed in a 1-L eggplant-shape flask, and 750 mlof hexane was added thereto. The resulting mixture was stirred with aglass rod until the hexane turned pale yellow, and thereafter themixture was heated at 85° C. in an oil bath. One hour after thebeginning of heating, the heating was terminated, and thereafter, theresulting product was filtered with No. 2 filter paper manufactured byADVANTEC.

Thereafter, the resulting filtrate was further subjected to suctionfiltration. The filtrate was allowed to stand in a draft for 24 hours toevaporate the hexane, thereby drying the resulting soybean dregs.

Next, a medium (composition: 2.0% by weight glucose, 1.0% by weightsucrose, 2.0% by weight soybean dregs, 0.5% by weight corn steep liquor,0.1% by weight K₂HPO₄, 0.05% by weight MgSO₄.7H₂O, 10 γ/ml, FeCl₂.6H₂O,pH 6.0) was prepared. The medium is a medium described in JapanesePatent Laid-Open No. Sho 60-156385.

To a 300-ml Erlenmeyer flask containing 30 ml of the above-mentionedmedium was added 0.2 ml of the culture (liquid medium) obtained in (1)in Example 1 mentioned above. The mixture was cultured with shaking(shaking rate: 130 rotations/min.) in a thermostatic bath at 28° C. for4 days. Next, the resulting culture medium was added to a 2-L Erlenmeyerflask containing 300 ml of the above-mentioned medium, and thereafter,the mixture was cultured with shaking (shaking rate: 130 rotations/min.)for 4 days under room-temperature conditions of 20° to 25° C.

After the termination of culture, the culture medium was subjected tosuction filtration through No. 2 filter paper manufactured by ADVANTECto remove the cells. Thereafter, the resulting filtrate was centrifugedat 4° C. at 14,800 rpm (30,000×g) for 30 minutes, to give a supernatantas the culture. The resulting culture was preserved at 4° C.

(2) Change in Enzyme Activity According to Reaction pH Conditions

In the assay of the phenol oxidase-like activity, each of2,6-dimethoxyphenol and p-phenylenediamine was used as a substrate.Also, in the assay of the phenol oxidase-like activity, there were usedglycine-HCl buffer at a pH of 2.5 to 4.0, sodium acetate buffer at a pHof 4.0 to 5.5, sodium phosphate buffer at a pH of 5.5 to 7.5, Tris-HClbuffer at a pH of 7.5 to 8.5 and glycine-NaOH buffer at a pH of 8.5 to11.0. Incidentally, when p-phenylenediamine is used as a substrate, inthe assay of the phenol oxidase-like activity, the assay was alsocarried out using sodium citrate buffer at a pH of 3.0 to 7.0.

To a semimicrocuvette were added 0.20 ml of a 500 mM buffer mentionedabove, 0.65 ml of deionized distilled water, 0.10 ml of a 50 mMsubstrate solution mentioned above, and 0.05 ml of the culture withstirring. The change in absorbance at 470 nm from 0 to 30 seconds wasdetermined at room temperature with trade name: UV-2400 (manufactured byShimadzu Corporation). Incidentally, the phenol oxidase-like activitywas also assayed in the same manner for the culture of the presentinvention obtained in Example 1 mentioned above. These results are shownin FIG. 7.

As a result, it was shown that the case of the culture (panels A and Bin FIG. 7) obtained in the above (1) has an optimum pH on an acidic pHas compared to the case of the culture of the present invention (panelsC and D in FIG. 7) obtained in Example 1 even when using any of thesubstrates.

Therefore, it is found that the phenol oxidase-like activity of theculture of the present invention and the phenol oxidase-like activity ofthe culture obtained by culturing under the condition of a pH of 6.0.are evidently different from each other.

FORMULATION EXAMPLES

Formulation Examples of the staining composition according to thepresent invention will be shown hereinbelow. When the followingcomposition is applied to white hair, the white hair can be stained toobscure the white hair. Here, the amounts blended are expressed in % byweight.

Formulation Example 1 (Gel Type) p-Phenylenediamine 1.5 Resorcin 0.3m-Metaaminophenol 0.1 Culture of (2) of Example 1 0.1 Sodium Ascorbate1.0 Hydroxyethyl Cellulose 1.0 Citric Acid Proper AmountMonoethanolamine Adjusted to pH of 7 Purified Water Balance Total 100.0Formulation Example 2 (Cream Type) p-Phenylenediamine 1.0 p-Aminophenol0.8 m-Aminophenol 0.1 Cetanol 6.0 Culture of (2) of Example 1 0.05Polyoxyethylene(20) Cetyl Ether 4.0 Stearyltrimethylammonium Chloride1.0 L-Cysteine Hydrochloride 0.2 Citric Acid Proper AmountMonoethanolamine Adjusted to pH of 7 Purified Water Balance Total 100.0Formulation Example 3 (Cream Type) 5,6-Dihydroxyindoline 1.05,6-Dihydroxyindole-2-carboxylic Acid 0.5 o-Aminophenol 0.5 Ethanol 5.0Stearyl Alcohol 1.5 Culture of (2) of Example 1 0.2 Polyoxyethylene(40)Hydrogenated 3.0 Castor Oil Polyglycerol Fatty Acid Ester 4.0N-Acetylcysteine 0.1 Xanthane gum 0.5 ACCULIN (Registered Trademark) 220.1 Hydroxyethyl Cellulose 0.1 Monoisopropanolamine Proper AmountMonoethanolamine Proper Amount Purified Water Balance Total 100.0Formulation Example 4 (Aerosol Type) Toluene-2,5-diamine 1.5p-Aminophenol 0.2 Resorcin 0.1 m-Aminophenol 0.1 Polyoxyethylene (15)Cetyl Ether 2.0 Propylene Glycol 5.0 Sodium Sulfite 0.3 MonoethanolamineProper Amount Citric Acid Proper Amount Culture of (2) of Example 1 0.3Liquefied Petroleum Gas 4.0 Purified Water Balance Total 100.0

INDUSTRIAL APPLICABILITY

According to the culture of the present invention, there can be utilizedin staining of fiber and hair, bleaching of pulp and fiber, removal of aphenolic compound in waste liquor, degradation of endocrine disruptors,preparation of a phenol resin, production of artificial Japaneselacquer, improvement in woody properties and the like, which can becarried out inexpensively and conveniently. In addition, according tothe method for producing the culture according to the present invention,the above-mentioned culture can be obtained conveniently, inexpensivelyand in a large amount. Further, according to the staining method and thestaining composition of the present invention, a subject to be stainedcan be stained efficiently at a nearly neutral pH, inexpensively andconveniently, with any of various dyes, especially phenolic compounds,aminophenolic compounds, and phenylenediamine compounds withoutsignificant fluctuation in the optimum pH under external environmentalconditions. Therefore, the staining method and the staining compositionare useful in staining of fiber and hair.

1. A culture from a strain belonging to the genus Flammulina, whereinthe culture has phenol oxidase-like activity and at least one substratespecificity selected from the group consisting of {circle around (1)} to{circle around (5)}: {circle around (1)} catalyzing an oxidativedecolorization reaction [decolorization activity] of each of: Evans'Blue represented by the formula (I):

Acid Blue 80 represented by the formula (II):

Remazol Brilliant Blue R represented by the formula (III):

Acid Violet 17 represented by the formula (IV):

{circle around (2)} catalyzing oxidative degradation reaction [oxidativedegradation activity] for lignin; {circle around (3)} catalyzingoxidative polymerization reaction [oxidative polymerization activity] ofIndigo Carmine represented by the formula (V):

Natural Orange 6 represented by the formula (VI):

{circle around (4)} catalyzing oxidative coupling reaction [oxidativecoupling activity] of 4-aminoantipyrine with one kind of a compoundselected from the group consisting of phenolic compounds, aminophenoliccompounds, diaminophenolic compounds and heterocyclic compounds; and{circle around (5)} catalyzing direct oxidative reaction [directoxidation activity] of one kind of a compound selected from the groupconsisting of phenolic compounds, aminophenolic compounds,diaminophenolic compounds and heterocyclic compounds, wherein theculture is obtainable by culturing a strain belonging to the genusFlammulina under pH conditions exceeding a pH of 7, and removing hyphaefrom the resulting culture medium.
 2. The culture according to claim 1,wherein the strain belonging to the genus Flammulina is Flammulinavelutipes.
 3. The culture according to claim 2, wherein Flammulinavelutipes is Flammulina velutipes strain IFO
 30601. 4. A culture from astrain belonging to the genus Flammulina, wherein the culture isobtainable by culturing a strain belonging to the genus Flammulina underpH conditions exceeding a pH of 7, and removing hyphae from theresulting culture medium, wherein the culture has phenol oxidase-likeactivity.
 5. A method for producing the culture from a strain belongingto the genus Flammulina according to claim 1, characterized in that themethod comprises culturing a strain belonging to the genus Flammulinaunder pH conditions exceeding a pH of 7, and removing hyphae from theresulting culture medium, to give a culture.
 6. The method according toclaim 5, wherein the strain belonging to the genus Flammulina isFlammulina velutipes.
 7. The method according to claim 6, whereinFlammulina velutipes is Flammulina velutipes strain IFO
 30601. 8. Astaining method characterized in that the staining method comprisescontacting a subject to be stained with a dye in the presence of theculture of claim
 1. 9. A staining composition comprising the culture ofclaim 1.